PROJECT SUMMARY/ABSTRACT Recent studies have shown that the vascular smooth muscle cell (SMC) can ?de-differentiate? and undergo substantial ?phenotypic modulation? during atherogenesis. Novel lineage-tracing data revealed that a subset of these cells assume a ?stem-like? phenotype and undergo significant clonal expansion, likely potentiating plaque progression. Unfortunately, we do not yet know the mechanism by which these clonal populations arise, how they promote disease, or whether they can be targeted for translational purposes. The broad, long-term goal of this proposal is to precisely define how clonal expansion occurs and how to suppress it. In doing so, we will learn how to target the ?atherosclerosis stem cell? and develop novel therapies for heart disease. Using new preliminary data generated via single-cell RNA-seq analysis of multicolor lineage-tracer models, our team will test the hypothesis that ?stem-like? SMCs evade phagocytic removal (allowing them to undergo clonal expansion) and activate the complement cascade (causing them to exacerbate vascular inflammation). The project combines unique transgenic animals, highly specialized human biorepository specimens and new interdisciplinary collaborations. The program is focused on three broad efforts which will: 1. Determine if pro-phagocytic therapies can specifically prevent clonal expansion; 2. Determine whether the ?stem-like? SMC requires the classical complement cascade to promote disease; and 3. Determine if clonal SMC expansion also has translational relevance to human atherosclerosis. This proposal will provide important insights into the root causes of SMC plasticity and determine how programmed cell removal, or ?efferocytosis? (from Greek, meaning to carry the dead to the grave) contributes to atherosclerosis. Ultimately, discoveries made in this highly flexible R35 project will support the stated mission of the National Institutes of Health, provide tools for other HLBS investigators, train the next generation of investigators, and lead to the development of new translational therapies for patients with cardiovascular disease.